Recent studies using vertebrate systems have shown that steroid hormones, in concert with their receptor proteins, bind to DNA sequences near particular genes, modulating the activities of those gene. Ecdysone, the steroid hormone of insects, has been studied for many years, but the details of its action are as yet unknown. We propose to elucidate the mechanism of ecdysone action by identifying those DNA sequences which confer ecdysoneinducibility on particular genes (EREs) and by studying the molecules that bind to those sequences. This proposal focuses on Drosophila genes in order to facilitate the eventual genetic analysis of these regulatory pathways. The Eip28/29 and Eip40 genes are well-characterized Drosophila genes which are induced by ecdysonein several cell lines. Indirect tests suggest that they participate in the primary response of these cells to the steroid hormone. A novel situation exists at the Eip40 locus: both strands of the DNA are transcribed and both transcripts are induced during the early response to ecdysone. Remaining structural questions at the Eip40 locus will be resolved by standard procedures. These experiments should suggest whether RNA complementarity plays a regulatory role at this locus. EREs for all 3 transcription units will be mapped. Once mapped, the DNA elements will be used as probes to identify the proteins with which they interact, perhaps including the ecdysone receptor. Identification of these critical components of the response should permit the design of in vivo assays to probe their structure. Such assays might include tests of DNase and Sl hypersensitivity and in vivo footprinting. The developmental pattern of Eip gene expression in flies will be determined and will be interpreted in the light of the assays of critical structures just described. Finally, efforts will be made to uncover the functional roles of the Eip gene products. These experiments should enhance our understanding of ecdysone, of transcriptional regulation in eukaryotes, and of the relation between steroid hormones and other elements of the developmental regulatory apparatus.